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Sunday, October 28, 2007

Alright, this week i'll be sharing more on the analysis, excision and identification of proteins. Also, i'll touch a bit on the DRP group that me and Jiaxin were put in charge of. This weeks blog is more of a reflective entry rather than a factual one.

After 16 weeks of SIP/MP, our group, comprising of me, Jiaxin, Ming Boon and Shahirah, still have not gotten any concrete results for our tabulation. Furthermore, most of our raw data that have been tabulated requires these final pieces of concrete results as evidence that the entire 20 weeks that were spent and the two projects were a success and also provide credibility that the protocols involved in the projects are reproducible. With 4 more weeks left, we crammed all our analysis, protein spot excision and identification of the possible proteins that have been selected.

Protein spot analysisAfter extracting our proteins for both the cell membrane and secretome project, we ran a 2-D gel before scanning the gels to acquire the image of the protein spots. The gels are placed in an imager called the Pharos FX Plus which scans the gels that have been stained with SYPRO Ruby or CyDye. The software, Quantity One, was selected to acquire the image on the computer. After getting pictures of the gels on the computer, another software called the PDQuest is used to customize the picture to our wishes. With this software, we are also able to magnify the protein spots that we choose to view such that a 3-D image of the protein spot will be shown on the computer.

For our cell membrane protein project, gels that were run according to experiment numbers would be compared. For instance, Experiments 6, 7 and 8 were protein samples from a selected S. Maltophilia strain and Experiments 9, 10 and 11 were from a different strain. The gels containing protein samples from Exp 6, 7 and 8 would be compared while Exp 9, 10 and 11 would be compared. From there, the best gel in terms of resolution and spot quality would be chosen for spot excision. For the secretome project, a comparison would be done for gels which run the protein samples belonging to bacterium grown at the same temperature.

Protein spot excisionThe protein spots would be selected through the PDQuest software. The Shimadzu Xcise would be used for the excision of spots and mounting of protein samples on the MALDI plate for identification. Although the Xcise has many advantages, the main one to be it is the automated spot excision and processing machine, its major disadvantage is that it still is controlled by the operator. Afetr using the Xcise for the second time, i realized that although it is not as tedious as manual spot cutting, processing and mounting, the automated version is still quite tedious. For instance, the spots that have been selected in the PDQuest software has to be re-selected on the computer sonnected to the Xcise machine. Manual calibration has to be done before selecting the spots and following the selection, the programming of the Xcise machine has to be done. Normally, after programming most machines, the operator would be free to perform other tasks. However, with the Xcise, we still have to monitor the automated process as there have been experiences whereby the excised protein spots were not cut properly or the excised protein spots were emptied into the wrong well. Thus, monitoring of the process is still required.

After the excision, processing and mounting, the protein samples would be mounted on the MALDI plate. The MALDI plate is then analyzed by a MALDI TOF/TOF which allows the sequencing of the peptides in the samples that have been spotted on the MALDI plate. From there, all the data has to be carefully tabulated.

After sharing with everyone the work process, I am sure that people will realize the research is never monotonous work and it requires a whole lot of effort from the individual. Also, a lot of thought has to be put in to actually organize all the data that has been established in such a way that would make sense to the readers.

DRP studentsFor our SIP assignment, we were also suppose to supervise a group of Differential Research Programme students that wereput under our care. Since i have actually performed the experiments that they were suppose to be performing to extract their own proteins, i thought the students would have actually been easy to handle. I was proven wrong.

In the first 2 weeks of them joining the programme, Jiaxin was left to handle the students since i was carrying out some experiments of our own. On the very first day i actually supervised them, the students were not even sure of the steps involved in their protocol. After a full day in the lab, they realized that they had actually performed the extraction wrongly. A few days later, they ran a 1D gel without denaturing their protein samples. Also, i realized that there was very little communication within the group to the extent that the individual performing the intial inoculation does not know the amount of cells that the individual performing the second inoculation has aliquoted. Also, although they kept their log book updated, they can not recall what they did only a few hours before. This could mean that they were actually following the protocol blindly. Thus, i conclude that if anyone so does wish to go into research, the individual should be completely interested in what he or she is doing.

Thus, i have come to the end of my blog. Although this is not a factual entry, i am still open to any questions. And thank you for listening to my complaints. HAHAHA.... See you guys in school soon.

Saturday, October 20, 2007

heya.......first and foremost i would like to wish all muslim frenz SELAMAT HARI RAYA.......dun forget to collect as many green packets as possible..it might be our last chance...hehehe....aniwei this week i would post something that was shown to me on the 1st day of work......ISOLATION OF PLASMA AND WBC..yah i noe..a bit outdated....

Now2 dun get confuse...yes i am under research but my lab is also involve in clinical stuff....soo yeah it's a mixture of both...so don't get a shock if u hear people in my lab go...quick sequence this first....the person is waiting for drugs to be administered......so back to the story.....

This whole thing is called blood processing and it is done by the research officer...we attachment students are not allowed to do this because our subsequent experiments will depend on this and if we screw up the blood processing....we are soo dead....we can't possibly ask the kind subject who 'donated' his/her blood to us to 'donate' again can we......

Phlebotomist will help us to draw blood from the volunteers (healthy individuals/patients that agreed to take part in our research). Then our counterpart in the clinical trials dept will then deliver the blood to us (the blood is collected in 3 EDTA tube-purple cap). Depending on what study the blood is for....the apropriate components will be isolated.....it is usually plasma, WBC and DNA..unfortunately i'm unable to post up on DNA isolation because we are not shown on tt....perhaps this wk they'll show us........well they have been promising to show us but the time has not come yet......now..after the delivery of blood......the show begins....

A) Isolation of plasma1. Centrifuge the 3 tubes of blood at 2000 rpm for 10 min-this separates the plasma from the other blood components2. Pipette the supernatant (plasma) into the 1.5ml eppendorf tubes.-pipette as much supernatant as possible but do not disturb the pellet3. Store the eppendorf tubes at -80oC fridge**the plasma is used for HPLC experiment to determine the drug level before, during and after infusion to study the drug pharmacodynamics and kinetics. thus plasma is usually collected for studies that involve drugs (usually in diseased subjects/patients to see their response)

B) Isolation of WBC1.Transfer 3ml of the pellet (from isolation of plasma) into 15 ml tubes-the pellet is actually RBC2. Add 9ml of RBC lysis solution to the tubes-this forms 1:3 blood to lysis solution ratio3. Incubate the tubes at room temperature for 10 min4. Centrifuge the tubes at 2000 rpm at 25oC for 10 min5. Pour away the supernatant6. Add 1ml of RBC lysis solution and pipette up and down7. Transfer everything from the tubes to eppendorf tubes8.Centrifuge the tubes at 3000rpm for 3min at 4oC9.Centrifuge at 15000rpm for 5 min at 4oCDiscard the supernatant11. Store the pellet (WBC) at -80oC fridge**this WBC will be used to isolate the DNA...soo die2 WBC will be collected for any studies since our lab is a very DNA lab....

tt's all for now......unfortunately this is my last post...left 3 more wks of SIP......enjoy it okies and let's fight till the end..............wishing everybody...ALL THE BEST FOR OUR SIP/MP......:D

Wednesday, October 17, 2007

Gomennasai, for the late posting! Here's my posting for week 16th. Basically, for the past few weeks I have been involved in the statistics analysis for both of my gene projects using the SPSS software. SPSS is the abbreviation used for Statistical Package for the Social Sciences; it provides almost everything and anything you required to perform an analysis.

Common Statistics Perform in SPSSIn SPSS, it comprises of multiple statistics tests, most of them are covered in year 1 math stats module. Two of the more commonly features used are the descriptive statistics and the bivariate/multivariate statistics.

Descriptive statisticsThe most fundamental and frequent statistic feature used. It summarized the samples’ results and portrays the tabulated results in forms of graphs or tables. Thence it is mainly used for quick and basic analysis. Here are some of the statistics tests used in descriptive statistics:FrequenciesA simple measurement used to computes/determine the mean, median, modes and SD of the results (variables).

Cross tabulationA cross tab provide information on 2 or more variables’ distribution consecutively and is usually presents a table format. Therefore, a cross tab is different from a frequency test. Some of the statistics tests used abide by with cross tab are chi-square (which test for clinical significant in variables), contingency coefficient (which test for strong interaction between variables) and phi coefficient (which test for degree of interaction between variables).

Bivariate/Multivariate StatisticsAs the name implied, this feature is only suitable for 2 variables. Any statistics involving more variables would be under the multivariate statistics. Similarly here is a list of the some of the statistics tests used in bivariate statistics:

t-testCommonly used when the sample size is minute or when the standard deviation is unknown. It is used to test if a null hypothesis is true or false (accept or reject). For example, if null hypothesis used is: mean of A = mean of B (where both the means of the variables is equal). After a series of calculation if t < a =" mean">

ANOVAAn ANOVA test compares the variables’ mean with their variances. Unlike t-test, ANOVA used variances instead of t value and also assess on more than two variables together.

So how and wat statistics should be applied to different data analysis?The answer would depend on wat type of “story “ you wish to express in your research. For instance, if you wish to find out the relationship of drug X on blood glucose level. Then a linear regression approach would be an ideal.

A linear regression is a technique used to determine the relationship of dependent variables with independent variables. In this case, blood glucose level is your dependent variable while the dosage of drug X would be the independent variables.Once you identify your variables you can start analyzed using both the descriptive statistics and the bivariate/multivariate statistics.

Another technique used for data analysis is called the factors analysis. This approach look into dependent variables, meanwhile, indirectly identifies the independent variables within. For example, by looking at blood glucose level (dependent variables), other independent variable such as concentration of drug X would be determined.

Lost in this statistics info?? Don’t worry maybe these definitions can help out.Dependent variablesA dependent variable is an outcome, a prediction that can be influenced by the independent variable. Usually is something, which cannot be control.

Independent variablesAn independent variable is something, which you may control or predict in experiment.

MeansMean equal average

Null hypothesisA null hypothesis is a presumed statement before statistics analysisData analysisFor SPSS to analysis a data/ samples results, a syntax is often used. Simply type in the code (instructions) you want the SPSS to do and select run. Subsequently, the results will appear in the output file in a graph, table, histogram formats (etc). Based on the tabulated results, interpretations can be made.

That all for this week!! Stay tune to next week SIP sharing!!If you have any question on my post feel free to leave comment.

Monday, October 1, 2007

Anyway, I am currently extracting RNA from Formalin-fixed, Paraffin-embedded blocks. Hence, I shall talk about this process today yup!

Reason for extraction/isolation of RNA : Using this RNA extracted from the blocks, I will carry out Reverse-Transcription PCR to reverse-transcribe the mRNA I have gotten into cDNA and then carry out Real-time PCR to look at the genetic expression of a particular gene I am interested in. For more info on Real-time PCR, do refer to my group member, Avery’s blog entry (July 22, 07) as I believe she had written a very detailed entry about this technique =))

RNA isolation must be carried out over 2 days.DAY 1 :(Sectioning)

Using the microtome, I will section about 45um of tissue sections into a eppendorf tube.

(Deparaffinissation)

Add 800uL of xylene into the tube and mix.Significance: Since I am using FFPE blocks, I will first have to remove the wax from the section .

Blot the tube briefly onto a paper towel to get rid of the ethanol residues.

Dry the tissue pellet for 10mins at 55ºC

(Overnight Proteinase K Digestion Incubation)

Add 100uL of Tissue Lysis Buffer, 16uL 10% SDS and 40uL proteinase K into the dried tubes and vortex briefly before incubating the tube overnight at 55ºC.Significance: This is to disrupt the protein structures in order to get the mRNA we want

DAY 2:

(RNA isolation)

Add 325uL of binding buffer and abs ethanol to the tube and pipette to mix.

Significance: Binding buffer will bind to the nucleic acids we want and allow the contaminants to flow through the filter and into the collection for discard.

Combine the filter tube and collection tube and pipette the lysate into the upper reservoir

Significance: The nucleic acids will bound to the chaotropic salts specifically to the surface of the glass fibers pre-packed in the filter tube while all other contaminants (salts, proteins and other cellular contaminants) will be washed off the column.

Centrifuge the High Pure filter for 2 mins at max speed.

Place the High Pure filter tube into a fresh 1.5ml reaction tube, add 90uL Elution Buffer and centrifuge for 1 min @ 8000rpm before collecting the flowthrough.

Add 10uL DNase Incubation Buffer and 1.0uL DNase I to the eluate and mix. Incubate for 45mins at 37 ºC

Significance: DNase I will help to remove any residual DNA since all we want is the RNA from the blocks.

Significance: A second incubation step with Proteinase K is to improve on the quality of the RNA as well as to ensure that all protein structures are disrupted.

Repeat the steps involved for RNA ISOLATION until the part in which the High Pure filter is centrifuged at max speed for 2 mins.

This time round, add only 50uL of Elution Buffer and centrifuge for 1 min@ 8000rpm.

Using the NanoDrop spectrophotometer, we can then check the RNA concentration of the 50uL eluate we obtained from our RNA extraction! =)

All the reagents/buffer were from this kit known as High Pure RNA Paraffin Kit from Roche, Switzerland. Therefore, i do not know the content of some of the reagents such as the Wash Buffer I and II which is why i cannot specifically tell you guys what each and every one of the reagents does ya. Hope this is alright with you guys. But i have tried to explain as many steps as possible so the whole idea behind this RNA isolation process should be pretty clear =)

(1) During sectioning, the tissue blocks are constantly exposed to the existence of RNase in the surrounding. Hence, the RNA in these blocks might risk getting destroyed during the sectioning process and affecting the total concentration of the RNA after the RNA extraction is done by the end of Day 2.(2) The process of fixing the tissue sample and embedding it in paraffin has caused severe degradation of the RNA. Therefore, it’s more difficult to isolate good quality RNA from FFPE tissues.(3) The “older” FFPE blocks tend to give poorer RNA concentration as compared to the tissues that were fixed only recently.

Ways to overcome these difficulties:

(1) Use RNase Zap to help reduce the amount of RNase that could be present in the surrounding (microtome/gloves). If talking is necessary during the isolation process, one could wear facemask so as to prevent the RNase in the saliva from degrading the samples.(2) Since it’s more difficult to isolate RNA from FFPE tissues, more sections could be collected in several tubes and pooled together so as to maximize the concentration of RNA to be collected after 2 days of extraction.(3) If given a choice, choose tissue blocks that were fixed and embedded recently (within 5 yrs) because studies have found that RNA in the blocks that were more than 10 year-old degrades significantly.